Polypeptide growth factors are important mediators of intercellular communication (Rubin et al. (1989), Proc. Natl. Acad. Sci. USA, 86:802-806). These molecules are generally released by one cell type and act to influence proliferation of other cell types.
One family of growth factors is the fibroblast growth factors (FGF). There are currently eight known FGF family members which share a relatedness among primary structures: basic fibroblast growth factor, bFGF (Abraham et al. (1986), EMBO J., 5:2523-2528); acidic fibroblast growth factor, aFGF (Jaye et al. (1986), Science, 233:541-545); int-2 gene product, int-2 (Dickson & Peters (1987), Nature, 326:833); hst/kFGF (Delli-Bovi et al. (1987), Cell, 50:729-737, and Yoshida et al. (1987), Proc. Natl. Acad. Sci. USA, 84:7305-7309); FGF-5 (Zhan et al. (1988), Mol. Cell. Biol., 8:3487-3495); FGF-6 (Marics et al. (1989), Oncogene, 4:335-340); keratinocyte growth factor (Finch et al. (1989), Science, 24:752-755; Rubin et al. (1989), Proc. Natl. Acad. Sci. USA, 86:802-806; Ron et al. (1993), The Journal of Biological Chemistry, 268(4):2984-2988; and Yan et al. (1991), In Vitro Cell. Dev. Biol., 27A:437-438); and hisactophilin (Habazzettl et al. (1992), Nature, 359:855-858).
Among the FGF family of proteins, keratinocyte growth factor (KGF) is a unique effector of non-fibroblast epithelial (particularly keratinocyte) cell proliferation derived from mesenchymal tissues. The term "native KGF" refers to a natural human (hKGF) or recombinant (rKGF) polypeptide (with or without a signal sequence) as depicted by the amino acid sequence presented in SEQ ID NO:2 or an allelic variant thereof. [Unless otherwise indicated, amino acid numbering for molecules described herein shall correspond to that presented for the mature form of the native molecule (i.e., minus the signal sequence), as depicted by amino acids 32 to 194 of SEQ ID NO:2, with the initial MET in each such sequence being considered residue number "0".]
Native KGF may be isolated from natural sources. For example, hKGF can be isolated from medium conditioned by an embryonic lung fibroblast cell line (Rubin et al. (1989), supra. Three chromatographic steps, namely heparin-Sepharose.TM. (Pharmacia, Piscataway, N.J.) affinity chromatography, HPLC gel filtration, and reverse-phase HPLC, were used to obtain a purified hKGF preparation. Approximately 6 mg of hKGF were recovered from 10 liters of conditioned medium. These chromatographic steps only recovered 0.8% total hKGF based upon a mitogenic activity assay. A further example teaches the use of another chromatographic step using heparin-Sepharose.TM. affinity and Mono-S.TM. ion-exchange chromatographys (Pharmacia, Piscataway, N.J.) for isolation of rKGF produced in bacteria (Ron et al. (1993), Journal of Biological Chemistry, 268:2984-2988).
The properties of keratinocyte growth factors suggest a potential for the application thereof as a drug for promoting specific stimulation of epithelial cell growth. It therefore would be desirable to develop a method or methods for obtaining relatively high levels of homogeneous keratinocyte growth factors to provide sufficient quantities of material for comprehensive in vitro and in vivo biological evaluation and for a potential therapeutic application.
It is the object of this invention to provide a novel method for the purification of keratinocyte growth factors.